Novel molecular triggers underlie valproate-induced liver injury and its alleviation by the omega-3 fatty acid DHA: role of inflammation and apoptosis.

BACKGROUND/AIM: Hepatic injury is a hallmark adverse reaction to Valproate (VPA), a common used drug in the management of numerous CNS disorders, including epilepsy. DHA has a myriad of health benefits, including renal- and hepato-protective effects. Unfortunately, however, the underpinnings of such liver-pertinent VPA- and DHA-actions remain largely undefined. Accordingly, this study attempted to unveil the cellular and molecular triggers whereby VPA evokes, while DHA abates, hepatotoxicity. METHODS: We evaluated activity and/or expression of cellular markers of oxidative stress, inflammation, and apoptosis in rat liver, following treatment with VPA (500 mg/kg/day) with and without concurrent treatment with DHA (250 mg/kg/day) for two weeks. RESULTS AND CONCLUSION: VPA promoted hepatic oxidative stress as evidenced by enhancing activity/expression of NADPH-oxidase and its subunits, a ROS-generator, and by accumulation of lipid-peroxides. Moreover, VPA enhanced hepatic phosphorylation/activation of mitogen-activated protein kinase (MAPK), and expression of cyclooxygenase-2(COX-2), as proinflammatory signals. Besides, VPA promoted hepatocellular apoptosis, as attested by enhanced expression of cleaved caspase-9 and increased number of TUNEL-positive hepatocytes. Lastly, VPA upregulated levels of hypoxia-inducible factor-1-alpha (HIF-1α), a multifaceted modulator of hepatocytic biology, and activity of its downstream antioxidant enzyme heme-oxygenase-1(HO-1). These changes were significantly blunted by co-administration of DHA. Our findings demonstrate that VPA activated NADPH-oxidase and HIF-1α to induce oxidative-stress and hypoxia as initiators of hepatic injury. These changes were further aggravated by up-regulation of inflammatory (MAPK and COX-2) and apoptotic cascades, but could be partly lessened by HO-1 activation. Concurrent administration of DHA mitigated all VPA-induced anomalies.

Valproate-induced liver injury: modulation by the omega-3 fatty acid DHA proposes a novel anticonvulsant regimen.

BACKGROUND: The polyunsaturated, ω-3 fatty acid, docosahexaenoic acid (DHA), claims diverse cytoprotective potentials, although via largely undefined triggers. Thus, we currently first tested the ability of DHA to ameliorate valproate (VPA)-evoked hepatotoxicity, to modulate its anticonvulsant effects, then sought the cellular and molecular basis of such actions. Lastly, we also verified whether DHA may kinetically alter plasma levels/clearance rate of VPA. METHODS AND RESULTS: VPA (500 mg/kg orally for 14 days in rats) evoked prominent hepatotoxicity that appeared as a marked rise (2- to 4-fold) in serum hepatic enzymes (γ-glutamyl transferase [γ-GT], alanine aminotransferase [ALT], and alkaline phosphatase [ALP]), increased hepatic lipid peroxide (LPO) and tumor necrosis factor-alpha (TNFα) levels, as well as myeloperoxidase (MPO) activity (3- to 5-fold), lowering of serum albumin (40 %), and depletion of liver reduced-glutathione (GSH, 35 %). Likewise, histopathologic examination revealed hepatocellular degeneration, replacement by inflammatory cells, focal pericentral necrosis, and micro/macrovesicular steatosis. Concurrent treatment with DHA (250 mg/kg) markedly blunted the elevated levels of liver enzymes, lipid peroxides, TNFα, and MPO activity, while raising serum albumin and hepatic GSH levels. DHA also alleviated most of the cytologic insults linked to VPA. Besides, in a pentylenetetrazole (PTZ) mouse convulsion model, DHA (250 mg/kg) markedly increased the latency in convulsion evoked by VPA, beyond their individual responses. Lastly, pharmacokinetic studies revealed that joint DHA administration did not alter serum VPA concentrations. CONCLUSIONS: DHA substantially ameliorated liver injury induced by VPA, while also markedly boosted its pharmacologic effects. DHA manipulated definite cellular machinery to curb liver oxidative stress and inflammation, without affecting VPA plasma levels. Collectively, these protective and synergy profiles for DHA propose a superior VPA-drug combination regimen.

Novel role of curcumin combined with bone marrow transplantation in reversing experimental diabetes: Effects on pancreatic islet regeneration, oxidative stress, and inflammatory cytokines.

Therapeutic utility of bone marrow transplantation in diabetes is an attractive approach. However, the oxidative stress generated by hyperglycemia may hinder ß-cell regeneration. The present study was undertaken to investigate the therapeutic potential of curcumin, a dietary spice with antioxidant activity, bone marrow transplantation, and their combined effects in the reversal of experimental diabetes. Diabetes was induced in mice by multiple low doses of streptozotocin. After the onset of diabetes, mice were treated with curcumin (10 mM; 100 µl/mouse, i.p., for 28 days) or received a single bone marrow transplantation (10(6) un-fractionated bone marrow cells), or both. Parameters of diabetes, integrity of pancreatic islets, pancreatic oxidative stress markers, and serum pro-inflammatory cytokines, were evaluated. Treatment with either curcumin or bone marrow transplantation significantly reversed streptozotocin-induced hyperglycemia/glucose intolerance, hypoinsulinemia, and damage of pancreatic islets. Interestingly, combination of curcumin and bone marrow transplantation elicited the most profound alleviation of such streptozotocin-evoked anomalies; including islet regeneration/insulin secretion. On the other hand, curcumin, either alone or combined with bone marrow transplantation, blunted the pancreatic lipid-peroxidation, up-regulated activities of the antioxidant enzymes, and suppressed serum levels of TNF-α and IL-1ß. Curcumin and single bone marrow transplantation proved their therapeutic potential in reversing diabetes when used in combination. Curcumin, via its antioxidant and anti-inflammatory effects, evidently enhanced the ability of bone marrow transplantation to regenerate functional pancreatic islets. Hence, the use of natural antioxidants combined with other therapeutic regimens to induce pancreatic regeneration is a promising strategy in the management of diabetes.

Resveratrol reverses hydrogen peroxide-induced proliferative effects in human coronary smooth muscle cells: a novel signaling mechanism.

BACKGROUND: In human coronary smooth muscle cells (HCSMCs), we tested the proatherogenic/proliferative potential of the reactive oxygen species (ROS), hydrogen peroxide (HP), and the ability of the polyphenol stilbene resveratrol (RSVL) to protect against such effects. METHODS: Activity for ERK1/2 and the kinase-G cascade were determined and correlated with HCSMC count before and after treatment with HP and/or RSVL. RESULTS: HP evoked concentration-dependent cell proliferation and stimulated ERK1/2 phosphorylation at active sites. Pretreatment with the MEK-ERK inhibitor (PD98059) reversed these effects of HP. RSVL (1-100 microM) elicited more prominent inhibition of HP-evoked cell proliferation and ERK1/2 activation. In addition, RSVL markedly enhanced cGMP formation, a response that was insensitive to the soluble guanylyl-cyclase (sGC) inhibitor (ODQ, 10 microM) but was obliterated with the phorbol ester, (PMA, 0.1 microM), a desensitizer of the pGC enzyme. Likewise, the RSVL-evoked cytostatic and ERK inhibitory effects were significantly reversed by the kinase-G-inhibitor, KT-5823 (10 microM). CONCLUSIONS: Collectively, RSVL activates the kinase-G system to counteract HP-induced ERK1/2 activation and coronary arterial proliferation. These effects for RSVL remain functional in endothelium-disrupted arteries, scenarios that commonly occur in advanced coronary heart disease.

Progesterone inhibition of MDM2 p90 protein in MCF-7 human breast cancer cell line is dependent on p53 levels.

The mdm2 gene encodes several protein isoforms with different molecular weights (p90, p80, p76 and p57). MDM2 p90 (usually considered to be the major MDM2 protein) binds to and inactivates P53. We have recently shown that growth inhibition of MCF-7 human breast cancer cells by progesterone is associated with P53 down-regulation. In this work, we analyzed the expression pattern of MDM2 proteins in three human breast cancer cell lines by western blotting with anti-MDM2 antibodies. We found a prominent expression of MDM2 p57 protein in cell lines which have non-functional P53 protein (T47D and MDA-MB-231) as compared to the p90, p80 isoforms, whereas p90 was the major protein isoform in MCF-7 cells that contain functional P53 protein. When MCF-7 cells were treated with 100 nM of progesterone, MDM2 p90 was inhibited but the highly expressed MDM2 p57 isoform was not. The inhibition of MDM2 p90 protein by progesterone was abrogated in MCF-7 cells transfected with a P53 expressing vector. To our knowledge, this is the first report linking progesterone-induced growth inhibition with down-regulation of the MDM2 protein. We present evidence that reestablishing of P53 expression by transient transfection of P53 cDNA in these cells enhances the expression level of MDM2 p90 isoform. The data indicate that expression of MDM2 p90 is regulated through a P53-dependent pathway in response to progesterone.

Differential recognition of resveratrol isomers by the human estrogen receptor-alpha: molecular dynamics evidence for stereoselective ligand binding.

Resveratrol (RSVL) is a phytoestrogen that occurs naturally in two forms (trans- (E) and cis- (Z)). We have conducted molecular dynamics (MD) studies to differentially characterize the estrogen receptor-alpha (ER-alpha) binding profiles of RSVL stereoisomers. Favorable orientations for RSVL isomers at the ER-alpha pocket were first inferred from (1) alignment with pharmacophoric elements of the pure ER-alpha agonists estradiol (E2) and (2) assessment of ligand recognition by the ER-alpha binding domain. Subsequently, these orientations for RSVL isomers were subjected to MD analyses versus E2. A 100-picosecond MD simulation revealed that E2 contributed four stable hydrogen bonds with the key ER-alpha pocket residue: Arg394, Glu353, His524, and Leu525. Further, E2 displayed favorable binding energy, conformational energy change (DeltaE), and movement of the binding pocket residues (RMSd). Compared to E2, (E)-RSVL lacked a hydrogen bond (HB) with His524 but formed three additional bonds with Gly521, Phe404, and Met343 of the ER-alpha pocket. Further, (E)-RSVL conferred more favorable energy of interaction, less favorable DeltaE, but comparable RMSd values. In contrast, (Z)-RSVL orientations missed hydrogen bonding (HB) with His524 and Leu525, two essential ligand binding residues, and/or produced considerably less favorable-binding energy, -DeltaE, and -RMSd values than did (E)-RSVL. In conclusion, the present study demonstrates the utility of this MD model in distinguishing between RSVL stereoisomers. The weak binding of (Z)-RSVL by the human ER-alpha binding is congruent with its inferior ligand profiles in ER-endowed biological systems. Further, evidence is provided for a considerable variation in the mode of recognition of the mixed agonist/antagonist (E)-RSVL, and the pure agonist E2.

Resveratrol activates adenylyl-cyclase in human breast cancer cells: a novel, estrogen receptor-independent cytostatic mechanism.

Resveratrol (RSVL) is a well-established chemopreventive agent in human breast cancer models. The molecular basis of its action is far less characterized. We investigated the effects of RSVL on activity of adenylate- and guanylate-cyclase (AC, GC) enzymes; two known cytostatic cascades in MCF-7 breast cancer cells. RSVL increased cAMP levels in both time- and concentration-dependent manners (t(1/2), 6.2 min; EC(50) 0.8 micro M). In contrast, it had no effect on cGMP levels. The stimulatory effects for RSVL on AC were not altered either by the protein synthesis inhibitor (actinomycin-D, 5 micro M) or the estrogen-receptor (ER) blockers (tamoxifen and ICI182,780, 1 micro M each). Likewise, cAMP formation by RSVL was insensitive to either the broad-spectrum phosphodiesterase (PDE) inhibitor (IBMX, 0.5 mM) or the cAMP-specific PDE inhibitor (rolipram, 10 micro M). Instead, these PDE inhibitors significantly augmented maximal cAMP formation by RSVL. Parallel experiments showed that either RSVL or rolipram inhibited the proliferation of these cells in a concentration-responsive manner. Further, concurrent treatment with RSVL and rolipram significantly enhanced their individual cytotoxic responses. The antiproliferative effects were appreciably reversed by the kinase-A inhibitors, Rp-cAMPS (100-300 micro M) or KT-5720 (10 micro M). Pretreatment with the cPLA(2) inhibitor arachidonyl trifluoromethyl ketone (10 micro M) markedly antagonized the cytotoxic effects of RSVL, but had no effect on that of rolipram. Altogether, the present study demonstrates, for the first time, that the chemotherapeutic agent RSVL is an agonist for the cAMP/kinase-A system, a documented pro-apoptic and cell-cycle suppressor in breast cancer cells.

Recognition of resveratrol by the human estrogen receptor-alpha: a molecular modeling approach to understand its biological actions.

OBJECTIVES: Resveratrol (RSVL) is an edible phytoestrogen with multifaceted health benefits that may originate from binding to the estrogen receptors. Despite its structural similarity to the estrogen receptor-alpha (ER alpha) agonist diethylstilbestrol (DES), RSVL showed distinct biological profiles in estrogen-responsive biological systems. The molecular basis of such biological profiles has been undefined. METHODS: We considered possible orientations for RSVL in ER alpha binding pocket. These conformations have been analyzed based on: (i) alignment with the key pharmacophoric elements of DES; (ii) computational energy of interaction, and (iii) pattern of accommodation at the ER alpha binding pocket. The characteristics of the most favored RSVL orientation have been compared with those of DES. RESULTS: Both RSVL and DES interacted with the catalytic amino acid triad of the ER alpha pocket (His524, Arg394 and Glu 353). However, unlike the Er alpha agonists DES and estradiol (E2), RSVL formed three additional hydrogen bonds with Gly521 and Leu525, two paramount ligand recognition residues, and with Met343 at the ER alpha binding cavity. Lastly, RSVL displayed a more favorable energy of interaction with the ER alpha binding cavity. CONCLUSIONS: The present study suggests, for the first time, that RSVL is well recognized by the human ER alpha but in a manner distinct from the pure agonists DES and E2. These variations may well entail the unique biological responses of RSVL in ER-responsive systems.

Resveratrol activates membrane-bound guanylyl cyclase in coronary arterial smooth muscle: a novel signaling mechanism in support of coronary protection.

Resveratrol (RSVL), an edible polyphenolic stilbene, claims a myriad of cardiovascular benefits. However, the molecular underpinnings of such actions are poorly understood. Currently, in sheep coronary arteries (SCA), RSVL markedly (threefold) enhanced cGMP formation (t(1/2): 6.5 min; EC(50): 3 microM). This response was not abrogated by the phosphodiesterase inhibitor (IBMX, 0.5 mM), but was partly sensitive (20-30%) to either removal of the endothelium, treatment with the nitric oxide synthase-inhibitor (L-NMMA, 10 microM), or with the soluble GC (sGC)-inhibitor (ODQ, 10 microM). In membrane preparations from denuded SCA, either RSVL or the pGC agonist atrial natriuretic peptide (ANP, 0.1-1 microM) activated GC in the particulate, but not in the soluble, membrane fraction. By contrast, the nitric oxide donor, sodium nitroprusside (SNP, 1-10 microM), stimulated GC only in the soluble fraction. Further, pretreatment with RSVL partly desensitized the ANP response, but was additive to that of SNP. In arterial tension studies, RSVL relaxed PGF(2alpha)-precontracted denuded rings in a concentration-dependent manner, a response that was markedly enhanced (approximately 18 fold) in the presence of IBMX. Conversely, precontraction with phorbol ester, which also desensitizes pGC, blunted relaxations to RSVL but not to forskolin or SNP. These findings demonstrate that RSVL increases cGMP in coronary arteries, mostly by activation of pGC. This pathway triggers vasorelaxant responses that remain effective in endothelium-disrupted arteries.

Endothelium-independent effect of estrogen on Ca(2+)-activated K(+) channels in human coronary artery smooth muscle cells.

OBJECTIVE: Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular/molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. METHODS: Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. RESULTS: Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.5 +/- 3 pS), calcium- and voltage-activated potassium (BK(Ca)) channel was the target of estrogen action. Channel activity was stimulated approximately 15-fold by nanomolar concentrations of 17 beta-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17 beta-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by inhibiting NO synthase with 10 microM N(G)-monomethyl-L-arginine. CONCLUSIONS: Our cellular and molecular studies identify the BK(Ca) channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BK(Ca) channel or a closely associated regulatory molecule, and further evidence suggests involvement of the NO/cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.